Tobacco Having Altered Amounts of Environmental Contaminants and Methods for Producing Such Lines

ABSTRACT

Disclosed herein are genetically modified tobacco plants, and tobacco products derived from such plants, comprising an altered uptake and/or altered levels of at least one environmental contaminant or other non-natural chemical as compared to an unmodified parent tobacco plant from which the modified plant is derived. The alteration in uptake and/or levels may be a reduction, or alternatively an increase, in the uptake and/or levels of the environmental contaminant or other non-natural chemical. These methods and plants are useful for improving tobacco products or in generating tobacco lines that may have environmentally useful properties.

RELATED APPLICATIONS

The present application is a continuation application and claimspriority to pending U.S. patent application Ser. No. 13/368,797, filedFeb. 8, 2012, entitled “Tobacco Having Altered Amounts of EnvironmentalContaminants and Methods For Producing Such Lines.” The disclosure ofU.S. patent application Ser. No. 13/368,797 is incorporated by referencein its entirety herein.

FIELD OF THE INVENTION

The present invention relates methods for making tobacco having alteredamounts of at least one environmental contaminant, such as a heavymetal, and tobacco lines produced by such methods.

BACKGROUND OF THE INVENTION

Popular smoking articles, such as cigarettes, may have a substantiallycylindrical rod shaped structure and include a charge, roll or column ofsmokeable material such as shredded tobacco (e.g., in cut filler form)surrounded by a paper wrapper. Also, tobacco may be used for smokelessproducts such as chewing tobacco and other smokeless tobacco products.For example, various types of smokeless tobacco products are set forthin U.S. Patent Publication No. 2011/0048434 the disclosure of which isincorporated by reference herein in its entirety.

Throughout the years, various methods have been developed to improve theflavor of tobacco and tobacco products. In addition to improving flavor,it would be helpful to produce tobacco plants, and tobacco productsderived from such plants, having reduced amounts of certain potentiallytoxic chemicals. For example, it would be of interest to produce tobaccoplants and tobacco products having reduced heavy metals.

Cadmium (Cd) is a heavy metal that can be found in soils. In the field,phosphate fertilizers can be a source of cadmium and the concentrationcan vary based on agricultural practices, soil characteristics, climateand plant varieties (Lugon-Moulin et al., Agronomy for SustainableDevelopment, 26:151-156, 2006). Cadmium can accumulate in tobacco.Studies indicate that stalk position, crop year and growing region mayhave an effect on cadmium content. Also, due to differing climates andsoils, establishment of true genetic differences in existing cultivarsmay be difficult to characterize with precision (Lugon-Moulin et al.,Chemosphere, 63: 1074-1086, 2006). For example, studies indicate thatpotential cadmium accumulation differences may exist between theNicotiana species but less so within varieties of Nicotiana tabacum(Wagner and Yeargan, Plant Physiology, 82: 274-279, 1986).

Since tobacco (e.g., lamina, stems, flowers and roots) is used in themanufacturing of cigarettes and alternative tobacco products, developingnew cultivars that less effectively translocate heavy metals such ascadmium from roots to shoots may be beneficial.

Also, as tobacco can take up such environmental contaminants, tobaccolines that take up an increased amount of such compounds may be used todecrease the amount of such chemicals in soil (e.g., for soil clean-up).

Thus, there is a general need for methods that can provide plant linescomprising altered uptake of a heavy metal contaminant or othernon-natural chemicals, or other compounds from the environment, wheresuch compounds may be potentially toxic if ingested over long periods oftime. The method should be designed so that even for plant species suchas tobacco that have a complex genome and thus require screening of alarge number of mutation events to isolate the mutation of interest, andthat are large and thus require extensive facilities for breeding,screening for the desired phenotype is economical.

SUMMARY OF THE INVENTION

The present invention provides tobacco plants having significantlyaltered uptake and/or altered levels of at least one environmentalcontaminant or other non-natural chemicals and methods of making suchplants.

In certain embodiments, the present invention provides methods of makingtobacco plants or a portion thereof having significantly altered uptakeand/or altered levels of at least one environmental contaminant or othernon-natural chemicals as compared to an unmodified tobacco plant orportion thereof from which the modified plant is derived. The alterationin uptake and/or levels may be a reduction in the uptake and/or levelsof the at least one environmental contaminant or other non-naturalchemicals as compared to an unmodified tobacco plant or portion thereoffrom which the modified plant is derived. Or, the alteration in uptakeand/or levels may be an increase in the uptake and/or levels of the atleast one environmental contaminant or other non-natural chemicals ascompared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived.

In certain embodiments, the method for producing a modified tobaccoplant or a portion thereof comprising altered uptake and/or alteredlevels of at least one environmental contaminant or other non-naturalchemical as compared to an unmodified tobacco plant or portion thereoffrom which the modified plant is derived may comprise the steps of:incubating seeds from the unmodified tobacco plant in a solutioncomprising a mutagen; washing the seeds free of the mutagen; germinatingthe seeds and growing M0 tobacco seedlings in the presence of aselection agent to generate at least one M0 tobacco plant comprising M1tobacco seeds, wherein the M1 tobacco seeds comprise at least onemutagenized M1 tobacco seed; and germinating the at least onemutagenized M1 tobacco seed to select for a modified M1 tobacco plant ora portion thereof comprising altered uptake and/or altered levels of atleast one environmental contaminant or other non-natural chemical. Incertain embodiments, the method may comprise germinating the M1 plants(and/or M2 and/or M3 and/or M4 plants and/or subsequent generations)derived from the M0 plants in a medium comprising a selection agent.

In yet other embodiments, the invention comprises a modified tobaccoplant or a portion thereof comprising altered uptake and/or alteredlevels of at least one environmental contaminant or other non-naturalchemical as compared to an unmodified tobacco plant or portion thereoffrom which the modified plant is derived. The alteration in uptakeand/or levels may be a reduction in the uptake and/or levels of the atleast one environmental contaminant or other non-natural chemicals ascompared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived. Or, the alteration in uptake and/orlevels may be an increase in the uptake and/or levels of the at leastone environmental contaminant or other non-natural chemicals as comparedto an unmodified tobacco plant or portion thereof from which themodified plant is derived.

In certain embodiments, the present invention comprises a tobacco plantderived from one of the tobacco lines described herein. For example, incertain embodiments, the invention may comprise a tobacco plantcomprising at least one of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or10TN-287-4 lines.

Also, in some embodiments, the present invention comprises tobaccoproducts made from the plants and tobacco lines of the invention. Forexample, in certain embodiments, the present invention comprises tobaccoproducts comprising a modified tobacco having decreased uptake and/orreduced levels of at least one environmental contaminant or othernon-natural chemical as compared to tobacco from an unmodified tobaccoplant or portion thereof from which the modified plant is derived.

In certain embodiments, the present invention comprises a composition(e.g., a tobacco product) comprising tobacco derived from one of thetobacco lines described herein. For example, in certain embodiments, theinvention may comprise a composition comprising at least one of the10TN-278-2, 10TN-253-4, 10TN-256-1 or 1-TN-287-4 lines.

In each of the embodiments of the invention, the environmentalcontaminant or other non-natural chemical having altered uptake maycomprise a heavy metal. The heavy metal may be at least one of arsenic(As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), selenium(Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag). In anembodiment, the heavy metal having altered uptake and/or levels iscadmium. Or, other heavy metals and/or other selected elements may bereduced.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features, aspects, and advantages of the present invention willbecome more apparent with reference to the following description,appended claims, and accompanying drawings.

FIG. 1 is a schematic representation illustrating a method forgenerating tobacco lines with altered levels of at least oneenvironmental contaminant or other non-natural chemical according to oneembodiment of the invention.

FIG. 2 shows the distribution of Cd in individual flue-cured M1 breedingline selections by plant tissue sample according to one embodiment ofthe invention. Bars in the graph are segmented in the order as presentedin the key on the right side of the graph.

DETAILED DESCRIPTION Definitions

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Also the terms “seed(s)” and “seedling(s)” includesingle and plural referents.

Reference to “dry weight percent” or “dry weight basis” refers to weighton the basis of dry ingredients (i.e., all ingredients except water).

The term “non-natural” refers to compounds that are not naturallyingested by animals and/or taken up and/or synthesized in the plantbeing modified by the methods of the invention (e.g., tobacco). Suchcompounds may be undesirable for human consumption. In some cases,however, such compounds may be beneficial for animal and/or humanconsumption.

The term “environmental contaminant” refers to a compound in theenvironment that is not normally found in the environment, or that isgenerally found in significantly lower levels in an uncontaminatedenvironment than in an environment that is considered to be contaminatedwith the compound. In some cases, the environmental contaminant may be aheavy metal. Or, the environmental contaminant may be another compoundthat has potentially harmful or non-beneficial effects when ingested byanimals and/or humans.

As defined herein, media may comprise compositions which are suited formaintenance or growth of biological tissue. Media may comprise water,buffered solutions, agar, or a growth medium, such as, but not limitedto, the media described in the examples herein. Generally, anycomposition which is biologically compatible with the plant of interestmay be suitable for use as a media or part of a media.

Also as defined herein, mutagenesis comprises a process that results ina modification of a DNA sequence. The term “mutagenesis” refers to theuse of a mutagenic agent to induce genetic mutations within a populationof individuals. A population to be mutagenized can comprise plants,parts of plants, or seeds. For mutagenized populations, the dosage ofthe mutagenic chemical or radiation can be determined experimentally foreach type of plant tissue such that a mutation frequency that is below athreshold level characterized by lethality or reproductive sterility isobtained. The number of M1 generation seeds or the size of M1 plantpopulations resulting from the mutagenic treatments can be estimatedbased upon the expected frequency of mutations.

The types of mutations that may be induced in a gene include, forexample, point mutations, additions, deletions, insertions,duplications, transitions, transversions, and/or inversions. Mutagenesismay cause frameshifts, or crosslinking of nucleotides, as well asmodification/substitution of bases such that binding of proteins to DNA(e.g. transcription factors) is altered. Or, mutations may cause othersequence based alterations of structure or function. Also included arehybrids made from such mutants as well as interspecific andintraspecific crosses.

In addition to the methods described in detail herein, in someembodiments, mutagenesis may be induced by growing plant cells in tissueculture, which can result in the production of somaclonal variants.Alternatively, application of standard protoplast culture methodologiesdeveloped for production of hybrid plants using protoplast fusion isalso useful for generating plants having variant gene expression.Accordingly, protoplasts may be generated from a first and a secondplant having variant gene expression. Calli may be cultured fromsuccessful protoplast fusions and plants regenerated. Resulting progenyhybrid plants may be identified and selected for variant gene expressionaccording to methods described herein and may be used in a breedingprotocols described herein. Also included are methods comprising geneticengineering such as site-directed mutagenesis.

The plants included in the plants of the invention also include plants(and/or lines derived from such plants) which may be geneticallyengineered using DNA from a different species (e.g., non-tobacco DNA fora tobacco plant) at a locus distinct from the genes relating to uptakeof environmental contaminant or other non-natural chemicals. Forexample, plants which are genetically engineered to be resistant topesticides and which are also modified by mutagenesis of tobacco genomicDNA to exhibit reduced uptake of at least one environmental contaminantor other non-natural chemical are included in the present invention. Inone embodiment, the plants of the present invention comprise plantshaving essentially the same antibiotic resistance profile as theunmodified plant parent line, such that the modified plants do notcomprise a novel antibiotic resistance as compared to the unmodifiedparent line.

A mutagen is defined as a substance (or treatment) which can change(mutate) the DNA in a cell. Suitable mutagenic agents include, forexample, chemical mutagens and ionizing radiation. Typical chemicalmutagens include, but are not limited to, ethyl methanesulfonate (EMS),nitrous acid, 5-bromouracil, methyl-nitrosoguanidine, sodium azide,acridine orange, ethidium bromide and frameshift mutagens such asproflavin and the like. Mutations can also be generated by radiation,such as UV, X-rays, γ-rays, fast neutron irradiation, and the like.Mutagens also include genetic elements such as viral vectors,transponsons, and the like, which can facilitate the insertion offoreign DNA into the tobacco genome.

Generally, the first generation treated with a mutagen comprises the M0(or M₀) generation. Subsequent generations are then described as M1 (orM₁) (i.e., one generation after the mutagenesis event), M2 (twogenerations after the mutagenesis event) and the like. Thus, as usedherein, M0 refers to plant cells (and plants grown therefrom) exposed toa mutagenic agent, while M1 refers to seeds produced by self-pollinatedM0 plants, and plants grown from such seeds. M2 is the progeny (seedsand plants) of self-pollinated M1 plants, M3 is the progeny ofself-pollinated M2 plants, M4 is the progeny of self-pollinated M3plants, M5 is the progeny of self-pollinated M4 plants, and so forth.Thus, the Mn+1 (or M_(n+1)) generation each the progeny ofself-pollinated plants of the previous Mn (or M_(n)) generation.

As used herein, a “modified plant”, or “modified tobacco plant” or“modified tobacco” includes plants, tobacco plants and tobacco that isgenetically modified (i.e., mutated) so as to have a different genotypeand phenotype than the unmodified plant (e.g., tobacco) from which themodified plant is derived.

As used herein, the “unmodified plant from which the modified plant isderived” refers to the parent plant line used to generate mutant (i.e.,“modified”) plant lines. As used herein, the M0 generation would be theunmodified plant from which the modified M1 plant is derived.

As defined herein, the physical appearance of an organism comprises itsphenotype, whereas the genetic composition of an organism comprises itsgenotype. Heterozygotes are defined as genomes which have differentalleles (i.e., DNA sequences) at a locus of interest. For example, aheterozygous mutation would be a plant having a mutated sequence at onlyone allele. Thus, heterozygotes have two distinct alleles for a gene,each of which can be passed to the next generation. Homozygotes aredefined as organisms having identical alleles at one or more loci. Thus,homozygotes carry the same alleles (e.g. two mutations or two normalsequences) at a locus of interest and, therefore, identical alleles willbe passed to all progeny.

Tobacco Having Altered Environmental Contaminants

The present invention provides methods of making tobacco plants havingsignificantly altered uptake and/or levels of at least one environmentalcontaminant or other non-natural chemicals. In certain embodiments, theinvention comprises methods for producing a modified tobacco plant or aportion thereof comprising generating a tobacco plant comprisingsignificantly altered uptake and/or altered levels of environmentalcontaminant or other non-natural chemicals as compared to an unmodifiedtobacco plant or a portion thereof from which the modified plant isderived.

In certain embodiments, the present invention provides methods of makingtobacco plants or a portion thereof (or tobacco lines derived from suchplants) having a decreased uptake and/or reduced levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived. In certain embodiments, the invention comprisestobacco plants and/or tobacco products made from plants of the inventionthat have a decreased uptake and/or reduced levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived.

In other embodiments, the present invention provides methods of makingtobacco plants or a portion thereof having significantly increaseduptake and/or increased levels of at least one environmental contaminantor other non-natural chemicals as compared to an unmodified tobaccoplant or portion thereof from which the modified plant is derived. Incertain embodiments, the invention comprises tobacco plants, or tobaccolines derived from such plants, or products made from such plants,having significantly increased uptake and/or increased levels of atleast one environmental contaminant or other non-natural chemical ascompared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived.

In certain embodiments of each of the methods, plants, plant lines, ortobacco products of the invention, the environmental contaminant orother non-natural chemical may comprise a heavy metal. The heavy metalhaving altered uptake and/or altered levels may be at least one ofarsenic (As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb),selenium (Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag). Forexample, in certain embodiments of each of the methods, plants, plantlines, or tobacco products of the invention the heavy metal havingaltered uptake and/or levels is cadmium. Or, other heavy metals and/orother selected elements may be altered in each of the methods, plants,plant lines, or tobacco products of the invention.

The present invention may be embodied in a variety of ways.

Methods of Making Tobacco Having Altered Environmental Contaminants

Embodiments of the present invention provide methods of making tobaccoplants or a portion thereof having altered uptake and/or altered levelsof at least one environmental contaminant or other non-natural chemicalas compared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived. The alteration in uptake and/or levelsmay be a reduction in the uptake and/or levels of the environmentalcontaminant or other non-natural chemical. Or, the alteration in uptakeand/or levels may be an increase in the uptake and/or levels of theenvironmental contaminant or other non-natural chemical.

In certain embodiments of each of the methods of making tobacco plantsor a portion thereof having altered uptake and/or altered levels of atleast one environmental contaminant or other non-natural chemical ascompared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived, the environmental contaminant or othernon-natural chemical may comprise a heavy metal. The heavy metal havingaltered uptake and/or altered levels may be at least one of arsenic(As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), selenium(Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag). In anembodiment, the heavy metal having altered uptake and/or levels iscadmium. Or, other heavy metals and/or other selected elements may havealtered uptake and/or levels.

In certain embodiments where alteration of uptake or levels of a heavymetal is desired, the selection agent is a heavy metal. The heavy metalmay be the same heavy metal for which alteration in uptake and/or levelsis desired. For example, in certain embodiments, where alteration ofcadmium uptake is desired, the selection agent is cadmium

Thus, in one embodiment, the present invention comprises a method forproducing a modified plant or a portion thereof (or a line derived fromthe plant) comprising an altered phenotype of altered uptake and/oraltered levels of at least one environmental contaminant or othernon-natural chemical as compared to an unmodified tobacco plant orportion thereof from which the modified plant is derived. The method maycomprise incubating at least one seed (i.e., a seed or seeds (seed(s)),for the plant of interest in a solution comprising a mutagen. The methodmay also comprise washing the at least one seed free of the mutagen. Themethod may additionally comprise germinating the at least one seed andgrowing M0 at least one seedling, i.e., a seedling or seedlings(seedling(s)). At this point, the method may comprise adding a selectionagent to the seedling(s), wherein the selection agent selects for achimeric M0 plant, wherein the M0 chimeric plant at least partiallycomprises the predetermined altered phenotype. The method may thencomprise growing the M0 plant to generate M1 seed(s), wherein the M1seed(s) comprise at least one mutagenized M1 seed comprising thepredetermined altered phenotype, and germinating the at least onemutagenized M1 seed to select for at least one M1 plant comprising thepredetermined altered phenotype. In certain embodiments, the method mayfurther comprise growing the at least one M1 plant to generate M2seed(s) and germinating the M2 seeds to grow M2 plants, wherein at leastone of the M2 plants is a homozygote for a mutation conferring theability to grow in the presence of the selection agent (e.g., an alteredphenotype and genotype). In certain embodiments, the method may comprisegerminating the M1 and/or M2 and/or M3 and/or M4 plants and/orsubsequent generations derived from the M0 plants in a medium comprisinga selection agent. Also in certain embodiments, the second mediumcomprising at least one selection agent is added to M0 seedlings atcertain developmental stages, as during a predetermined time periodafter germination.

For example, in certain embodiments, the method may comprise incubatingat least one tobacco seed from an unmodified tobacco plant in a solutioncomprising a mutagen; washing the at least one seed free of the mutagen;germinating the at least one seed and growing at least one M0 tobaccoseedlings in the presence of a selection agent to generate at least oneM0 tobacco plant comprising M1 tobacco seeds, wherein the M1 tobaccoseeds from the chimeric tobacco plant comprise at least one mutagenizedM1 tobacco seed; and germinating the at least one mutagenized M1 tobaccoseed to select for a modified M1 tobacco plant comprising significantlyaltered uptake and/or altered levels of environmental contaminant orother non-natural chemicals as compared to the unmodified tobacco plant.

In alternate embodiments, the modified plant (or a line derived from theplant) has at least a 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%decrease in at least one environmental contaminant or other non-naturalchemical. In alternate embodiments, the modified plant has at least a1.2, 1.5, 2, 4, 6, 8, 10, 20, 50 or 100-fold reduction in at least oneenvironmental contaminant or other non-natural chemical. For example, incertain embodiments, the modified tobacco plant comprises at least a100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% reduction in cadmiumlevels.

In alternate embodiments, the modified plant (or a line derived from theplant) has at least a 10, 20, 30, 40, 50, 100, 200 or 500% increase inat least one environmental contaminant or other non-natural chemical. Inalternate embodiments, the modified plant has at least a 1.2, 1.5, 2, 4,6, 8, 10, 20, 50 or 100-fold increase in at least one environmentalcontaminant or other non-natural chemical. For example, in certainembodiments, the modified tobacco plant comprises at least a 10, 20, 30,40, 50, 100, 200 or 500% increase in cadmium levels.

Also in certain embodiments of the methods of the invention, a mediumcomprising at least one selection agent is added to M0 seedlings atcertain developmental stages, as during a predetermined time periodafter germination. For tobacco (e.g., Nicotiana tabacum) a mediumcomprising at least one selection agent may be added to M0 seedlings 7to 14 days after germination. For example, the second medium comprisingat least one selection agent may be added to M0 seedlings about 10 daysafter germination. In alternate embodiments, the selection may becontinuously present during growth of the M0 seedlings.

As discussed further herein, a variety of plants may be modified usingthe methods of the invention. In certain embodiments, the plantcomprises an alloploid genome. In certain embodiments, the plant istobacco. In certain preferred embodiments, the tobacco is the genusNicotiana. More preferably, the tobacco may comprise the speciesNicotiana tabacum. Or, as described herein, any of the genus ofNicotiana thereof may be used. Such members are described in U.S. PatentPublication Nos. 2006/0185686 and 2011/0174323, the disclosures of whichare incorporated by reference in their entireties herein.

Or, other tobaccos as described in more detail herein may be used. Forexample, tobaccos used in the methods of the invention may include wildtobaccos or tobaccos that have been genetically modified in some manner.Thus, the tobaccos may include types of tobaccos such as flue-curedtobacco, burley tobacco, sun-cured tobacco (e.g., Oriental tobacco orIndian Kurnool), Maryland tobacco, dark tobacco, dark-fired tobacco,dark air cured (e.g., passanda, cubano, jatim and bezuki tobaccos) orlight air cured (e.g., North Wisconsin and galpao tobaccos), and Rusticatobaccos, as well as other rare or specialty tobaccos or even green oruncured tobaccos. Representative Oriental tobaccos include katerini,prelip, komotini, xanthi and gambol tobaccos.

In certain embodiments, the mutagen is ethyl methanesulfonate (EMS). Insome embodiments, the EMS comprises a final concentration of 0.01 to 2%.Or, the EMS may comprise a final concentration of 0.05 to 1%. Or, theEMS may comprise a final concentration of 0.1 to 0.7%. For example, incertain embodiments a concentration of 0.5% may be used. Or, otherchemical mutagens (or irradiation) may be used.

In certain embodiments, the mutagenized seeds are suspended withinsolidified agar plates containing the selection agent. In otherembodiments, seeds may be suspended in a nutrient medium and applied toa semi-permeable surface for growth prior to addition of the selectionagent. Or, selection may be performed using a hydroponic system or insoil. Or, other methods of in vitro selection known in the art may beused.

Embodiments of the present invention comprise methods for plantmutagenesis that includes a selection step at the M0 chimeric stage. Forexample, in embodiments of each of the methods, plants (plant lines) andproducts of the invention, the invention comprises selection of plantscomprising resistance to high (e.g., potentially lethal) amounts of aheavy metal as a selection agent. The resistance may be due to analteration in uptake and/or levels that comprises a reduction in theuptake and/or levels of the environmental contaminant or othernon-natural chemical. Or, the resistance may be due to an alteration inuptake and/or levels that comprises an increase in the uptake and/orlevels of the environmental contaminant or other non-natural chemical.

For example, in embodiments of each of the methods, plants (plant lines)and products of the invention, the invention comprises selection ofplants comprising resistance to high (e.g., potentially lethal) amountsof cadmium (Cd) as a selection agent. Or, other selection agents, e.g.,other heavy metal environmental contaminants or other non-naturalchemicals, may be used.

The concentration of the selection agent may vary depending upon thenature of the environment and the selection agent used. In someembodiments, where cadmium is the selection agent, the cadmium ispresent at a concentration ranging from about 0.02 to about 15 mM, or0.04 to about 10 mM, or about 0.050 to about 5 mM. Or, the cadmium maybe present at a concentration ranging from about 0.075 to about 1 mM.Or, the cadmium is present at a concentration ranging from 0.1 to about0.3 mM. Or, the cadmium selection agent may be present at about 0.15 mM.

The M0 plant will, in certain embodiments, comprise a subpopulation ofcells resistant to the selection agent; these cells may confer viabilityto the entire plant when the plant is grown in the presence of aselection agent such as, but not limited to, elevated levels of cadmium.The present invention thus can eliminate the growth of a largepopulation of the M0 plants for production of M1 seeds and therebysubstantially reduces the number of progeny that must be screened at theM1 stage, thus resulting in a cost-effective plant breeding programsuitable for large plants, or plants comprising complex genomes, such astobacco. Other methods used to produce N. tabacum lines comprising asignificantly increased amino acid content, and specifically, asignificant increase in threonine are described in U.S. Pat. Nos.6,730,832, 7,173,170 and 7,825,305, the disclosures of which areincorporated by reference in their entireties herein.

Embodiments of the invention may also comprise seeds derived frommodified plants that have been generated using the methods of theinvention, wherein the seeds are capable of propagating the modifiedplant lines having altered uptake and/or altered levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived. The alteration in uptake and/or levels may be areduction in the uptake and/or levels of the environmental contaminantor other non-natural chemical. Or, the alteration in uptake and/orlevels may be an increase in the uptake and/or levels of theenvironmental contaminant or other non-natural chemical.

In certain embodiments of the seeds made by the methods of theinvention, the environmental contaminant or other non-natural chemicalmay comprise a heavy metal. The heavy metal having an altered uptakeand/or altered levels may be at least one of arsenic (As), cadmium (Cd),chromium (Cr), nickel (Ni), lead (Pb), selenium (Se), zinc (Zn), copper(Cu), mercury (Hg) or silver (Ag). In an embodiment, the heavy metalhaving altered uptake and/or levels is cadmium. Or, other heavy metalsand/or other selected elements may be altered in seeds made by themethod of the invention.

Also, as discussed in more detail herein, the invention comprisestobacco products derived from plants and/or plant lines generated usingthe methods of the invention such as leaves for chewing tobacco,flue-cured leaves for smoking tobacco, and other known tobacco products.In certain embodiments, the present invention comprises methods foraltering the levels of at least one environmental contaminant ornon-natural compound that may be present in the tobacco and/or generatedupon heating the tobacco and/or improving the flavor of a tobaccoproduct, comprising generating a modified tobacco plant having altereduptake and/or altered levels of at least one environmental contaminantor non-natural chemical (or a compound derived therefrom by heating thetobacco), mixing the modified tobacco with unmodified tobacco, andincluding the mixture in a tobacco product.

Preferably, the modified tobacco plant is made by mutagenizing tobaccoseeds and selecting for at least partially mutagenized plants havingaltered uptake and/or altered levels of at least one environmentalcontaminant or other non-natural chemical as compared to an unmodifiedtobacco plant or portion thereof from which the modified plant isderived. The alteration in uptake and/or levels may be a reduction inthe uptake and/or levels of the environmental contaminant or othernon-natural chemical. Or, the alteration in uptake and/or levels may bean increase in the uptake and/or levels of the environmental contaminantor other non-natural chemical

Thus, embodiments the present invention relate to utilization of amethod for producing plants with an altered phenotype by selection atthe M0 chimeric stage and the use of this method to product plantshaving a decrease in at least one amino acid. Mutagenesis has been usedas a conventional breeding method to develop improved cultivars of anumber crops, including tobacco (see e.g. A. M. van Harten, MutationBreeding: Theory and Practical Applications, pp. 1-63, Cambridge Univ.Press, New York, N. Y., 1998). Generally, the target plant materialsused to develop desired mutant using chemical mutagens are classifiedinto two categories: (1) seed and (2) tissue or cell culture.

For example, seeds may be treated with specific mutagens, and thesurviving seeds grown to produce their progenies (e.g. Heremans andJacobs, 1995). The generation that grows from the mutagenized seed iscalled the M0 generation, and the progeny collected from the M0 plantsare the M1 generation, from which the desired mutants are usuallyselected. Further selection of plants which are homozygous for themutation of interest may be made by growing progeny from M1 plants (i.e.the M2 generation) under selective conditions.

Although mutagenesis and selection can be performed on tissue culturecells (e.g. Cattoir-Reynaerts et al., 1983; Dotson et al., 1990; Hibberdet al., 1980), the mutant cells or tissues must be regenerated tofertile plants. Establishment of a system for regeneration of a fertileplant from the genotype of interest can be time-consuming, expensive,and requires a high level of technical expertise. In addition, undesiredsomaclonal variation often occurs in regenerated mutants of interest asa result of autosomal chromosome duplications.

Genetic engineering has also been used produce transgenic plants. It hasbeen shown that expression of a bacterial heavy metal transporter inArabidopsis can enhance resistance to and decrease uptake of heavymetals (J. Lee et al., Plant Physiology, 2003, 133:589-596). Still, thistype of approach is technically demanding, requires introduction offoreign DNA into the genome, and generally does not generate the widevariety of mutants needed for propagation of a crop in variousecosystems. In addition, the effects of transgenic crops produced bygenetic engineering on the long-term stability of ecosystems is notknown (N. C. Ellstrand, 2001, Plant Physiol., 125: 1543-1545). Finally,transgenic crops have not been widely accepted by the public, as forexample, in certain European countries.

It is therefore an object of the present invention to utilize ascreening method whereby the screening is at least in part performedusing M0 plants. Referring now to FIG. 1, in one aspect, the inventionprovides a method of producing tobacco (e.g., Nicotiana tabacum) lineshaving an altered (i.e., increased or reduced) uptake and/or an alteredamount of at least one environmental contaminant or other non-naturalchemical. In an embodiment, the method comprises the steps of: (a)mutagenizing tobacco seeds 2 in a solution containing the mutagen ethylmethanesulfonate (EMS) 4 at a concentration of about 0.5% for 20 hrs;(b) washing the mutagenized seeds in water 6; (c) sterilizing the seeds2 with 70% ethanol followed by 20% Chlorox bleach 8 on an agitatingshaker 10; (d) rinsing with sterile water 6; (e) suspending the seeds 2in nutrient medium with 0.1% Agar (a semi-solid colloidal suspension)12; (f) applying about 0.750 ml of the suspension with about 50 seeds 2onto a solidified phytoagar plate containing a selection agent 16; (g)germinating the seeds and growing the seedlings 18 in a tissue cultureroom at 25° C. with a 16-h photoperiod from cool-white fluorescentlamps; (h) transplanting the healthy seedlings 18 to soil and growing asM0 plants 22 in a greenhouse; (i) planting individual M1 seeds 23 fromM0 plants 22 in a field to generate M1 plants 24; (j) testing the M1plants for altered levels of the environmental contaminant or othernon-natural chemical of interest (e.g., Cd); (k) growing M2 seeds 25from the M1 plants with an altered level of the environmentalcontaminant or other non-natural chemical of interest, and testing forM2 lines 26 producing altered levels of the environmental contaminant orother non-natural chemical of interest (e.g., Cd). These non-chimerictobacco plants may be a mixture of heterozygotes and homozygotes.Additional selection step may be added for selection of tobacco lines inthe field based on chemistry.

These modified plants and/or lines also provide the basis for theproduction of hybrid lines, utilizing as one or both parents, the novellines of the present invention. Also within the scope of the presentinvention are clones, somaclones, and derivatives of the novel lines.

In certain embodiments, the methods of the present invention are used togenerate a tobacco plant derived from one of the tobacco lines describedherein. For example, in certain embodiments, the methods of theinvention are used to generate a tobacco plant comprising at least oneof the 10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines. Or, theinvention may comprise methods to make other tobacco lines describedherein. Thus, in certain embodiments, the modified tobacco plants madeby the methods of the invention may comprise at least one of the10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines described herein,wherein a representative sample of seeds for these lines have beendeposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209), on Feb. 21, 2012, underconditions prescribed by the Budapest Treaty, and which will have allrestrictions on accessibility of the seeds irrevocably removed uponissuance of a patent. The deposited seeds have been tested anddesignated as viable as of Mar. 20, 2012, and assigned ATCC AccessionNumbers PTA-12602 (10TN-278-2), PTA-12601 (10TN-253-4), PTA-12599(10TN-256-1) and PTA-12600 (10TN-287-4), respectively.

Tobacco Plants Having Altered Levels of at Least One EnvironmentalContaminant

In certain embodiments, the invention comprises plants or a portionthereof having significantly altered uptake and/or altered levels of atleast one environmental contaminant or other non-natural chemical ascompared to an unmodified tobacco plant or portion thereof from whichthe modified plant is derived. In certain embodiments, the inventioncomprises a modified tobacco plant or a portion thereof comprising asignificantly altered uptake and/or altered levels of environmentalcontaminant or other non-natural chemicals as compared to an unmodifiedtobacco plant or a portion thereof from which the modified plant isderived, wherein the genome of the modified tobacco plant comprises amutation that exhibits a dominant phenotype of resistance to growth inthe presence of a selection agent.

The alteration in uptake and/or levels may be a reduction in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. Or, the alteration in uptake and/or levels may be an increasein the uptake and/or levels of the environmental contaminant or othernon-natural chemical.

In certain embodiments of each of the plants of the invention, theenvironmental contaminant or other non-natural chemical may comprise aheavy metal. The heavy metal having altered uptake and/or altered levelsmay be at least one of arsenic (As), cadmium (Cd), chromium (Cr), nickel(Ni), lead (Pb), selenium (Se), zinc (Zn), copper (Cu), mercury (Hg) orsilver (Ag). In an embodiment, the heavy metal having altered uptakeand/or levels is cadmium. Or, other heavy metals and/or other selectedelements may have altered uptake and/or levels in the plants of theinvention.

In certain embodiments where alteration of uptake or levels of a heavymetal is desired, the selection agent is a heavy metal. The heavy metalmay be the same heavy metal for which alteration in uptake and/or levelsis desired. For example, in certain embodiments, where alteration ofcadmium uptake is desired, the selection agent is cadmium.

As discussed further herein, a variety of plants may be modified. Incertain embodiments, the plant comprises an alloploid genome. In certainembodiments, the plant is tobacco. In certain preferred embodiments, thetobacco is the genus Nicotiana. More preferably, the tobacco maycomprise the species Nicotiana tabacum. Or, as described herein, any ofthe genus of Nicotiana may be used. Such tobaccos are described in U.S.Patent Publication Nos. 2006/0185686 and 2011/0174323, the disclosuresof which are incorporated by reference in their entireties herein.

Or, other tobaccos may be used. For example, tobaccos used to generatethe plants of the invention may include wild tobaccos or tobaccos thathave been genetically modified in some manner. Thus, the tobaccos mayinclude types of tobaccos such as flue-cured tobacco, burley tobacco,sun-cured tobacco (e.g., Oriental tobacco or Indian Kurnool), Marylandtobacco, dark tobacco, dark-fired tobacco, dark air cured (e.g.,passanda, cubano, jatim and bezuki tobaccos) or light air cured (e.g.,North Wisconsin and galpao tobaccos), and Rustica tobaccos, as well asother rare or specialty tobaccos or even green or uncured tobaccos.Representative Oriental tobaccos include katerini, prelip, komotini,xanthi and gambol tobaccos.

Thus, in certain embodiments, the present invention may comprise atobacco plant or a portion thereof comprising a modified tobacco havingsignificantly altered uptake and/or altered levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived. The alteration in uptake and/or levels may be areduction in the uptake and/or levels of the environmental contaminantor other non-natural chemical. Or, the alteration in uptake and/orlevels may be an increase in the uptake and/or levels of theenvironmental contaminant or other non-natural chemical.

For example, the present invention may comprise a tobacco plant or aportion thereof comprising a significantly altered uptake and/or alteredlevels of at least one environmental contaminant or other non-naturalchemical as compared to an unmodified tobacco or a portion thereofwherein the genome of the modified tobacco plant comprises an alloploidgenome having a mutation that exhibits a dominant phenotype ofresistance to growth in the presence of cadmium or another selectionagent.

The plants (e.g., tobacco plants) included in the present invention alsoinclude plants (and/or lines derived from such plants) which may begenetically engineered using foreign (e.g., non-tobacco) DNA at a locusdistinct from the genes relating to levels of the environmentalcontaminant or other non-natural chemical of interest in the tobacco(e.g., genes relating to uptake of the environmental contaminant orother non-natural chemical of interest). For example, tobacco plantswhich are genetically engineered to be resistant to pesticides and whichare also modified by mutagenesis of tobacco genomic DNA (e.g., at a generelating to uptake of the environmental contaminant or other non-naturalchemical of interest) to have a significantly altered uptake and/oraltered levels of at least one environmental contaminant or othernon-natural chemical as compared to an unmodified plant from which themodified plant is derived, are included in the present invention.

Also, embodiments of the present invention may comprise a modifiedtobacco plant or a portion thereof, or a tobacco line derived from theplant, having a significantly altered uptake and/or altered levels of atleast one environmental contaminant or other non-natural chemical ascompared to an unmodified parent tobacco plant or a portion thereofand/or tobacco line from which the modified plant is derived, whereinthe genome of the modified tobacco plant consists of, or consistsessentially of, plant DNA. Preferably, the genome of the modifiedtobacco plant consists of, or consists essentially of tobacco DNA.

The present invention also includes a modified tobacco plant or aportion thereof and/or a tobacco line derived from the plant havingsignificantly altered uptake and/or altered levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified parent tobacco line or a portion thereof, wherein themodified plant has consists of, or consists essentially of, the sameantibiotic resistance as the unmodified parent line.

In certain embodiments of each of the tobacco plants of the invention,the environmental contaminant or other non-natural chemical may comprisea heavy metal. The heavy metal having altered uptake and/or reducedlevels may be at least one of arsenic (As), cadmium (Cd), chromium (Cr),nickel (Ni), lead (Pb), selenium (Se), zinc (Zn), copper (Cu), mercury(Hg) or silver (Ag). In an embodiment, the heavy metal having altereduptake and/or levels is cadmium. Or, other heavy metals and/or otherselected elements may have altered uptake and/or levels.

Thus, in another aspect, the present invention comprises a modifiedtobacco plant or a portion thereof, or a tobacco line derived from theplant, having a significantly altered uptake and/or altered levels of atleast one environmental contaminant or other non-natural chemical ascompared to an unmodified parent tobacco plant, wherein the genome ofthe modified tobacco plant consists of, or consists essentially of,plant DNA, and/or wherein the modified tobacco plant is produced by thesteps of mutagenesis of tobacco genomic DNA and selection of M0 plantshaving a mutation of interest. More preferably, the gene or genes of themodified tobacco plant relating to the uptake and/or levels of theenvironmental contaminant or other non-natural chemical of interestconsist of, or consist essentially of tobacco genomic DNA. Thealteration in uptake and/or levels may be a reduction in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. Or, the alteration in uptake and/or levels may be an increasein the uptake and/or levels of the environmental contaminant or othernon-natural chemical.

Thus, in certain embodiments, the present invention comprises a modifiedtobacco plant and/or a tobacco line derived from the tobacco planthaving a significantly altered uptake and/or altered levels of at leastone environmental contaminant or other non-natural chemical as comparedto an unmodified parent tobacco line, wherein the tobacco plant isproduced by mutagenesis of tobacco genomic DNA and selection of M0plants having a mutation of interest. The alteration in uptake and/orlevels may be a reduction in the uptake and/or levels of theenvironmental contaminant or other non-natural chemical. Or, thealteration in uptake and/or levels may be an increase in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. In certain embodiments, the tobacco plant is produced by thesteps of: mutagenizing at least a tobacco seed(s); germinating themutagenized seed(s) in the presence of a selection agent e.g., cadmiumand/or another heavy metal for production of tobacco having alteredcadmium and/or other heavy metal uptake; growing at least one M0 plantin the presence of the selection agent to generate M1 seed(s), whereinthe M1 seed(s) comprise at least one mutagenized M1 seed; andgerminating the at least one mutagenized M1 seed in medium to select fora mutagenized M1 plants. Thus, in an embodiment, the modified tobaccoplant is made by a method comprising the steps of: incubating at leastone tobacco seed from an unmodified tobacco plant in a solutioncomprising a mutagen; washing the at least one seed free of the mutagen;germinating the at least one seed and growing at least one M0 tobaccoseedlings in the presence of a selection agent to generate at least oneM0 tobacco plant comprising M1 tobacco seeds, wherein the M1 tobaccoseeds from the chimeric tobacco plant comprise at least one mutagenizedM1 tobacco seed; and germinating the at least one mutagenized M1 tobaccoseed to select for a modified M1 tobacco plant comprising significantlyaltered uptake and/or altered levels of environmental contaminant orother non-natural chemicals as compared to the unmodified tobacco plant.In certain embodiments, the method of preparing the modified plantincludes the steps of growing the M1 tobacco plants to generate M2seed(s), and further germinating the M2 seeds to grow M2 plants, whereinat least one of the M2 plants is a homozygote for a mutation conferringthe ability to grow in the presence of increased a selection agent(e.g., cadmium). In certain embodiments, the method may comprisegerminating the M1 and/or M2 and/or M3 and/or M4 plants and/orsubsequent generations derived from the M0 plants in a medium (or soil)comprising a selection agent.

In alternate embodiments, the modified plant (or a line derived from theplant) has at least a 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%decrease in at least one environmental contaminant. In alternateembodiments, the modified plant has at least a 1.2, 1.5, 2, 4, 6, 8, 10,20, 50 or 100-fold reduction in at least one environmental contaminantor other non-natural chemical. For example, in certain embodiments, themodified tobacco plant comprises at least a 100%, 90%, 80%, 70%, 60%,50%, 40%, 30%, 20% or 10% reduction in cadmium levels.

In alternate embodiments, the modified plant (or a line derived from theplant) has at least a 10, 20, 30, 40, 50, 100, 200 or 500% increase inat least one environmental contaminant. In alternate embodiments, themodified plant has at least a 1.2, 1.5, 2, 4, 6, 8, 10, 20, 50 or100-fold increase in at least one environmental contaminant or othernon-natural chemical. For example, in certain embodiments, the modifiedtobacco plant comprises at least a 10, 20, 30, 40, 50, 100, 200 or 500%increase in cadmium levels.

Thus, in certain embodiments, the present invention provides tobaccoplants and/or lines derived from such plants that have been mutated soas to exhibit altered levels (i.e., concentration) of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived.

In certain embodiments, these novel lines have less than 0.5 micrograms(μg) cadmium per gram of dry weight of tobacco in flue cured leaf. Thismay represent about a 2-fold decrease in cadmium over the unmodified N.tabacum parent, which may normally yields a maximum of about 0.9-1.25 μgcadmium per milligram of dry weight of tobacco depending upon the field,time of year and/or other factors.

In other embodiments, these novel lines may uptake increase amounts ofcadmium so as to have about 1.49 or more micrograms (μg) cadmium pergram of dry weight of tobacco in cured leaf. This may represent about a20% increase in cadmium over unmodified N. tabacum parent, whichnormally yields a maximum of about 0.9-1.25 μg cadmium per milligram ofdry weight of tobacco. Such plants may be useful for reducing theamounts of cadmium in a field.

The absolute amount of a specific environmental contaminant or othernon-natural chemical may be dependent on processing of the leaf, or thedevelopmental stage of the plant.

In certain embodiments, the present invention comprises a tobacco plantderived from one of the tobacco lines described herein. For example, incertain embodiments, the invention may comprise a tobacco plantcomprising at least one of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or10TN-287-4 lines. Or, the invention may comprise other tobacco linesdescribed herein. Thus, in certain embodiments, the modified tobaccoplants made by the methods of the invention may comprise at least one ofthe 10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines describedherein, wherein a representative sample of seeds for these lines havebeen deposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209), on Feb. 21, 2012, underconditions prescribed by the Budapest Treaty, and which will have allrestrictions on accessibility of the seeds irrevocably removed uponissuance of a patent. The deposited seeds have been tested anddesignated as viable as of Mar. 20, 2012, and assigned ATCC AccessionNumbers PTA-12602 (10TN-278-2), PTA-12601 (10TN-253-4), PTA-12599(10TN-256-1) and PTA-12600 (10TN-287-4), respectively.

Tobacco Products

In certain embodiments, the invention comprises compositions and/ortobacco products made from tobacco plants or a portion thereof havingsignificantly altered uptake and/or reduced levels of at least oneenvironmental contaminant or other non-natural chemical as compared toan unmodified tobacco plant or portion thereof from which the modifiedplant is derived. In certain embodiments, the invention may comprise atobacco product comprising a modified tobacco having significantlyaltered uptake and/or altered levels of environmental contaminant ascompared to an unmodified tobacco from which the modified tobacco isderived.

For various embodiments of each of the products of the invention, thealteration in uptake and/or levels may be a reduction in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. Or, the alteration in uptake and/or levels may be an increasein the uptake and/or levels of the environmental contaminant or othernon-natural chemical if such chemicals are beneficial for consumption.

In certain embodiments of the various compositions and/or tobaccoproducts of the invention, the environmental contaminant or othernon-natural chemical may comprise a heavy metal. The heavy metal havingaltered uptake and/or altered levels may be at least one of arsenic(As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), selenium(Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag). In anembodiment, the heavy metal having altered uptake and/or levels iscadmium. Or, other heavy metals and/or other selected elements may havealtered uptake and/or levels. For example, in certain embodiments, thetobacco products of the invention may comprise a tobacco productcomprising a modified tobacco having reduced levels of cadmium ascompared to an unmodified tobacco. Or, other heavy metals or anotherenvironmental contaminant or other non-natural chemical may be reduced.

In certain embodiments where alteration of uptake or levels of a heavymetal is desired, the selection agent is a heavy metal. The heavy metalmay be the same heavy metal for which alteration in uptake and/or levelsis desired. For example, in certain embodiments, where alteration ofcadmium uptake is desired, the selection agent is cadmium.

Any of the modified plants described herein may be used in thecompositions and/or products of the invention. For example, the presentinvention may comprise a composition and/or tobacco product comprising amodified tobacco having altered uptake and/or altered levels of at leastone environmental contaminant or other non-natural chemical as comparedto an unmodified tobacco plant or portion thereof from which themodified plant is derived, wherein the genome of the modified tobaccoplant comprises an alloploid genome, and/or has been geneticallyengineered using foreign (e.g., non-tobacco) DNA at a locus distinctfrom the genes relating to levels of the environmental contaminant orother non-natural chemical of interest in the tobacco, and/or whereinthe genome of the modified tobacco plant consists of, or consistsessentially of, plant DNA, and/or wherein the modified plant hasconsists of, or consists essentially of, the same antibiotic resistanceas the unmodified parent line, and having a mutation that exhibits adominant phenotype of resistance to growth in the presence of aselection agent (e.g., cadmium or another selection agent). Thealteration in uptake and/or levels may be a reduction in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. Or, the alteration in uptake and/or levels may be an increasein the uptake and/or levels of the environmental contaminant or othernon-natural chemical.

In alternate embodiments, the composition and/or tobacco product has atleast a 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% decreased uptakeand/or reduced levels of at least one environmental contaminant or othernon-natural chemical as compared to non-modified tobacco. In alternateembodiments, the composition and/or tobacco product has at least a 1.2,1.5, 2, 4, 6, 8, 10, 20, 50 or 100-fold decreased uptake and/or reducedlevels of at least one environmental contaminant or other non-naturalchemical. For example, in certain embodiments, the modified tobaccoplant comprises at least a 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%or 10% reduction in cadmium levels.

In alternate embodiments, the composition and/or tobacco product has atleast a 10, 20, 30, 40, 50, 100, 200 or 500% increased uptake and/orincreased levels of at least one environmental contaminant or othernon-natural chemical as compared to non-modified tobacco. In alternateembodiments, the composition and/or tobacco product has at least a 1.2,1.5, 2, 4, 6, 8, 10, 20, 50 or 100-fold increased uptake and/orincreased levels of at least one environmental contaminant or othernon-natural chemical. For example, in certain embodiments, the modifiedtobacco plant comprises at least a 10, 20, 30, 40, 50, 100, 200 or 500%increase in cadmium levels.

As discussed further herein, a variety of tobacco plants may be modifiedto generate the compositions and/or products of the invention. Incertain embodiments, the plant comprises an alloploid genome. In certainembodiments, the plant is tobacco. In certain preferred embodiments, thetobacco is the genus Nicotiana. More preferably, the tobacco maycomprise the species Nicotiana tabacum. Or, as described herein, any ofthe genus of Nicotiana or blends thereof may be used. Such tobaccos andblends are described in U.S. Patent Publication Nos. 2006/0185686 and2011/0174323, the disclosures of which are incorporated by reference intheir entireties herein.

Or, other tobaccos and blends may be used. Tobaccos used in the productsof the invention may vary and generally include wild tobaccos ortobaccos that have been genetically modified in some manner. Forexample, the tobaccos may include types of tobaccos such as flue-curedtobacco, burley tobacco, sun-cured tobacco (e.g., Oriental tobacco orIndian Kurnool), Maryland tobacco, dark tobacco, dark-fired tobacco,dark air cured (e.g., passanda, cubano, jatim and bezuki tobaccos) orlight air cured (e.g., North Wisconsin and galpao tobaccos), and Rusticatobaccos, as well as other rare or specialty tobaccos or even green oruncured tobaccos. Representative Oriental tobaccos include katerini,prelip, komotini, xanthi and gambol tobaccos.

In certain embodiments of the invention, different parts of the plantand/or individual tobacco grades may be evaluated for the level of anenvironmental contaminant or other non-natural chemical and/or othernon-natural chemicals in smoke. For example, in certain embodiments,upper stalk grades may have less cadmium (or other non-naturalcontaminants or environmental contaminants) than lower stalk grades.Also, certain tobacco-specific grades (e.g., burley and oriental grades)may have reduced or increased levels of cadmium (or other non-naturalcontaminants or environmental contaminants) as compared to other grades(e.g., flue-cured grades).

In certain embodiments, the invention provides a heat-treated tobaccocomposition. As used herein, the term “heat-treated tobacco composition”refers to a composition comprising a tobacco material that has beenthermally processed at an elevated temperature, such as a temperature ofat least about 60° C., more typically at least about 100° C., for a timesufficient to alter the character or nature of the tobacco composition,for example, for as at least about 10 minutes. In some cases, the heattreatment process may alter the chemistry or sensory characteristics(e.g., taste and aroma) of the tobacco composition. The heat treatmentprocess can be a modified version of conventional tobacco treatmentprocesses, such as processes adapted to form flavorful and aromaticcompounds (e.g., Maillard reaction products), processes adapted forpasteurization of tobacco compositions, processes for preparing tobaccocasing products, reconstituted tobacco processes (e.g., cast sheet andpaper-making reconstituted tobacco processes), tobacco extractionprocesses, reordering processes, toasting processes, steam treatments,and drying processes.

Examples of tobacco products that may be generated using the tobaccoplants and/or tobacco lines of the present invention are described inU.S. Patent Publication No. US 2011/0048434, the disclosure of which isincorporated by reference in its entirety herein. Thus, the tobaccocompositions of the invention can be used as an additive for a smokingarticle, or as a smokeless tobacco composition, such as loose moistsnuff, loose dry snuff, chewing tobacco, pelletized tobacco pieces,tobacco comprising an insulated fuel element as described in U.S. Pat.No. 5,819,751 (i.e., heat not burn technology), extruded or formedtobacco strips, pieces, rods, or sticks, finely divided ground powders,finely divided or milled agglomerates of powdered pieces and components,flake-like pieces, molded processed tobacco pieces, pieces oftobacco-containing gum, rolls of tape-like films, readilywater-dissolvable or water-dispersible films or strips, or capsule-likematerials.

Tobaccos used in the invention, including tobacco compositions intendedto be used in a smokeless form as tobacco products of the invention, mayincorporate a single type of tobacco (e.g., in a so-called “straightgrade” form). For example, the tobacco within a tobacco composition maybe composed solely of flue-cured tobacco (e.g., all of the tobacco maybe composed, or derived from, either flue-cured tobacco lamina or amixture of flue-cured tobacco lamina and flue-cured tobacco stem). Thetobacco within a tobacco composition also may have a so-called “blended”form. For example, the tobacco within a tobacco composition of thepresent invention may include a mixture of parts or pieces offlue-cured, burley (e.g., Malawi burley tobacco) and Oriental tobaccos(e.g., as tobacco composed of, or derived from, tobacco lamina, or amixture of tobacco lamina and tobacco stem). For example, arepresentative blend may incorporate about 30 to about 70 parts burleytobacco (e.g., lamina, or lamina and stem), and about 30 to about 70parts flue cured tobacco (e.g., stem, lamina, or lamina and stem) on adry weight basis. Other exemplary tobacco blends may incorporate about75 parts flue-cured tobacco, about 15 parts burley tobacco, and about 10parts Oriental tobacco; or about 65 parts flue-cured tobacco, about 25parts burley tobacco, and about 10 parts Oriental tobacco; or about 65parts flue-cured tobacco, about 10 parts burley tobacco, and about 25parts Oriental tobacco; on a dry weight basis or similar percentages(e.g., within about a 10% range of each of the individual components fora total of 100%). Other exemplary tobacco blends incorporate about 20 toabout 30 parts Oriental tobacco and about 70 to about 80 partsflue-cured tobacco (e.g., within about a 10% range of each of theindividual components for a total of 100%). Or, other tobaccos ortobacco blends, such as those described in U.S. Patent Publication No.2006/0185686 the disclosure of which is incorporated by reference in itsentirety herein may be used. Or, other blends may be used.

The relative amount of tobacco within the composition and/or tobaccoproduct may vary. Preferably, the amount of tobacco within the tobaccoproduct ranges from at least about 10 percent or at least about 25percent, on a dry weight basis. In certain instances, the amounts ofother components within the tobacco product may exceed about 20, 30, 40,50, 60, 70, 80 or 90 percent, on a dry weight basis. A typical range oftobacco material in the tobacco product may range from about 10 to about60 weight percent, more often about 20 to about 40 weight percent on adry basis. For example, the tobacco product may include additionalflavorants, fillers, binders, buffering agents, colorants, andhumectants.

The tobacco products of the invention may be formulated as variousarticles of manufacture. For smokeless tobacco compositions, the tobaccocompositions of the invention can be formed into desired product shapes.The method and apparatus used to form the tobacco composition willdepend on the desired shape. For example, the tobacco composition canhave the form of compressed tobacco pellets, multi-layered extrudedpieces, extruded or formed rods or sticks, compositions having one typeof tobacco formulation surrounded by a different type of tobaccoformulation, rolls of tape-like films, readily water-dissolvable orwater-dispersible films or strips, or capsule-like materials possessingan outer shell (e.g., a pliable or hard outer shell that can be clear,colorless, translucent or highly colored in nature) and an inner regionpossessing tobacco or tobacco flavor.

The tobacco compositions of the invention may be useful as additives forthe manufacture of smoking articles. The tobacco of the invention can beincorporated into the tobacco blends, representative cigarettecomponents, and representative cigarettes manufactured therefrom. Forexample, the tobacco leaves of the present invention can be incorporatedinto a smoking article as part of the smokeable material charge. Or, thetobacco of the invention can be incorporated into a cigarette filter(e.g., in the filter plug, plug wrap, or tipping paper) or incorporatedinto cigarette wrapping paper, preferably on the inside surface, duringthe cigarette manufacturing process.

In certain embodiments of the compositions and/or tobacco products ofthe invention, the resulting smoking article is characterized by asignificantly altered levels of at least one environmental contaminantor non-natural chemical, or a compound derived therefrom, in mainstreamsmoke during use. For example, the smoking article can be characterizedby a significantly decreased level of at least one environmentalcontaminant or other non-natural chemical, or a compound derivedtherefrom, in mainstream smoke relative to an control smoking article(i.e., a comparable smoking article except containing unmodified tobaccoin place of the modified tobacco of the invention) of at least about 10percent, at least about 20 percent, at least about 30 percent, at leastabout 40 percent, at least about 50 percent, at least about 60 percent,at least about 70 percent, at least about 80 percent, or more. Thesmoking article of the invention, such as a cigarette, containing thetobacco composition of the invention can produce a significantlydecreased level of at least one environmental contaminant or othernon-natural chemical, or a compound derived therefrom, by weight inmainstream smoke as compared to a control smoking article smoked usingthe same smoking machine and under the same smoking conditions, such asthe smoking machines and smoking conditions set forth in ISO 3308:1991and ISO 4387:1991, which are incorporated by reference herein. Or, insome cases there may be an increase in a non-natural compound, or acompound derived therefrom, if such chemicals are beneficial forconsumption.

In certain embodiments, these novel lines have less than 0.5 micrograms(μg) cadmium per gram of dry weight of tobacco in cured leaf. This mayrepresent about a 2-fold decrease in cadmium over unmodified N. tabacumparent, which normally yields a maximum of about 0.33-2.24 μg/mg (seee.g., Lugon-Moulin et al., Chemosphere, 2006, 1074-1086), or about0.9-1.25 μg cadmium per milligram of dry weight of tobacco. In somecases (e.g., where sludge has been applied to a field, the level ofcadmium may be as high as 9.46 μg/mg (Adamu et al., EnvironmentalPollution, 1989, 56:113-126). The absolute amount of a specificenvironmental contaminant or other non-natural chemical may be dependenton the field, the time of year, processing of the leaf, or thedevelopmental stage of the plant, as well as other factors.

In certain embodiments, the present invention comprises a composition(e.g., a tobacco product) comprising tobacco derived from one of thetobacco lines described herein. For example, in certain embodiments, theinvention may comprise a composition comprising at least one of the10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines. Or, thecomposition may comprise other tobacco lines described herein. Thus, incertain embodiments, the composition and/or tobacco product comprisesmay comprise at least one of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or10TN-287-4 lines described herein, wherein a representative sample ofseeds for these lines have been deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209), onFeb. 21, 2012,_under conditions prescribed by the Budapest Treaty, andwhich will have all restrictions on accessibility of the seedsirrevocably removed upon issuance of a patent. The deposited seeds havebeen tested and designated as viable as of Mar. 20, 2012, and assignedATCC Accession Numbers PTA-12602 (10TN-278-2), PTA-12601 (10TN-253-4),PTA-12599 (10TN-256-1) and PTA-12600 (10TN-287-4), respectively.

The present invention may be better understood by reference to thefollowing non-limiting examples.

Example 1 Mutation of Tobacco

Seed from NL Madole, a dark-fired tobacco variety, and K326, aflue-cured variety, were mutagenized with 0.5% EMS (ethyl methanesulfonate) and designated as M0. The M0 mutant populations were screenedon tissue culture media containing 150 μM Cd.

To generate mutagenized tobacco, various (i.e., about 10 separate)aliquots of the NL Madole or K326 seeds were incubated in a solutioncontaining ethyl methane sulfonate (EMS) at a concentration of about0.5% for 20 hrs. Both K326 seeds and non-mutagenized NL Madole seedswere used as the control. The treated seeds were then washed with MiliQwater (purified with an NANO pure II system; Barnstead/Thermolyne Corp.;Dubuque, Iowa) for 30 minutes and sterilized with 70% ethanol for 30seconds followed by 20% Clorox for 20 min on an agitating shaker.

The M0 mutant populations were then screened on tissue culture mediacontaining 150 μM Cd. Thus, after rinsing with sterile MiliQ water atleast 5 times, the seeds (about 50 seeds per plate) were suspended insolidified phytoagar plates with ½ Murashige and Skoog Salt (MSS)medium+1.5% sucrose, 5 g phytoagar and selection agent (150 μM Cd). MSSmedium is described in Table 1 of U.S. Pat. No. 7,173,170; thedescription of this medium is incorporated by reference herein in itsentirety. The seeds were allowed to germinate in a tissue culture roomat 25° C. with a 16-h photoperiod using cool-white fluorescent lamps(Sylvania, Danvers, Mass.) with an intensity of approximately 80 μE m⁻²s⁻¹. After 10 days, the growth medium was removed and the same mediumcontaining 150 μM Cd, was added to the seedlings. The surviving plantswere then transplanted into soil and grown in a greenhouse fordevelopment of N. tabacum lines resistant to high levels of Cd.

Surviving M0 plantlets selected on Cd-rich media were rescued andtransferred to soil in a growth room. Later, these M0 plants weretransferred to the greenhouse and self-seed was collected from eachplant and designated as M1. M1 lines were grown under normal fieldconditions. Each M1 line was flue-cured according to traditionalpractices.

This M1 seed was rescreened by growing in the presence of the selectionagent (i.e., 150 μM Cd) using a tissue culture vertical plate techniquewhereby plantlets were grown vertically and shoots and roots weremeasured. Selections were based on fresh shoot weight and root length.Analysis of shoot and root data revealed significant differences amongthe M1 lines tested. Selected M1 plants were transferred to thegreenhouse and self seed was collected (M2) for further propagation.

Example 2 Analysis of for Cd Levels in Various Tobacco Lines MutatedUsing the Methods of the Invention

Twenty-two potential fields were evaluated and soil samples weresubmitted for Cd analysis. A field containing relatively high levels ofCd was identified in Greeneville, Tenn. (see Table 1). M1 and M2 tobaccobreeding lines were evaluated and screened under field conditions atthis location. In addition, several wild species of N. tabacum that havebeen reported to have resistance to Cd uptake were tested alongside M1and M2 selections for comparison.

All samples were collected at bud stage. Lower leaf lamina was collectedfrom individual M1 plants for analysis because of individual plantvariation at the M1 generation. Lower leaf lamina was collected fromwhole plots of a few of the M2 lines for analysis. All lamina was frozenon dry ice, and freeze-dried to maintain leaf integrity. Freeze-driedsamples were analyzed for content of Cd and other selected heavy metals.Table 1 below shows an analysis of acid (1N HNO₃) extracts of soil fromthe selected field for As, Cd, Cr, Ni, Pb and Se. Reported results arethe average and standard deviation of duplicate 1 N HNO3 extractscorrected to the soil dry weight.

TABLE 1 Metal Conc. (μg/g) Std. Dev. As 0.117 0.008 Cd 0.111 0.000 Cr2.700 0.200 Ni 1.540 0.040 Pb 7.800 0.100 Se 0.020 0.008

Four plants from the M1 lines were selected for generation advancementto M2, bagged to prevent outcrossing and treated with TRACER® or BELT®insecticide to prevent seed damage by insects. A single plant wasselected in each, and M2 line and M3 seed was produced as statedpreviously for the M₁ advancement. This field experiment was arandomized complete block design with three replicates. Data wasanalyzed using JMP® statistical software (α=0.05) (SAS, Cary, N.C.).

Flue-Cured Results:

Results for M1 lines are shown in Table 2.

Ninety-six experimental flue-cured M1 selections were evaluated againstK326 as the control (Table 2). It was found that twenty-four M1selections accumulated significantly more Cd than control, and fiftylines accumulated significantly less Cd than control. Table 2 shows meanCd content in individual flue-cured M1 breeding line selections. InTable 2, values for the control are shown in italics. Levels notconnected by the same letter or symbol are significantly different. TwoM1 selections, 10TN-269-2 and 10TN-266-2, shown in bold font in Table 2,accumulated less Cd than the GOTHIATEK® standard of 0.5 μg/g.

TABLE 2 Entry Pedigree/Name Mean Cd (μg/g) 10TN-290-1 EMS K326 (M₁)1.488 A 10TN-288-4 EMS K326 (M₁) 1.456 AB 10TN-288-1 EMS K326 (M₁) 1.452AB 10TN-281-1 EMS K326 (M₁) 1.414 AB 101-N-291-1 EMS K326 (M₁) 1.398 BC10TN-292-1 EMS K326 (M₁) 1.359 C 10TN-277-3 EMS K326 (M₁) 1.341 C10TN-290-2 EMS K326 (M₁) 1.256 D 10TN-293-2 EMS K326 (M₁) 1.179 E10TN-290-3 EMS K326 (M₁) 1.167 EF 10TN-286-1 EMS K326 (M₁) 1.163 EF10TN-266-3 EMS K326 (M₁) 1.155 EF 10TN-282-4 EMS K326 (M₁) 1.145 EF10TN-292-4 EMS K326 (M₁) 1.130 EF 10TN-282-1 EMS K326 (M₁) 1.115 EF10TN-291-4 EMS K326 (M₁) 1.109 EFG 10TN-291-2 EMS K326 (M₁) 1.096 FG10TN-290-4 EMS K326 (M₁) 1.036 GHI 10TN-293-1 EMS K326 (M₁) 1.030 HI10TN-277-1 EMS K326 (M₁) 1.020 IJK 10TN-282-3 EMS K326 (M₁) 1.009 IJKL10TN-280-1 EMS K326 (M₁) 1.004 IJKLM 10TN-277-2 EMS K326 (M₁) 1.004IJKLM 10TN-281-3 EMS K326 (M₁) 0.999 IJKLMN 10TN-286-4 EMS K326 (M₁)0.984 IJKLMNO 10TN-294-2 EMS K326 (M₁) 0.969 IJKLMNOP 10TN-267-1 EMSK326 (M₁) 0.957 JKLMNOPQ 10TN-274-3 EMS K326 (M₁) 0.956 JKLMNOPQ10TN-286-2 EMS K326 (M₁) 0.952 KLMNOPQR 10TN-274-1 EMS K326 (M₁) 0.947KLMNOPQRS 10TN-281-2 EMS K326 (M₁) 0.939 LMNOPQRST 10TN-2 K326 Control0.938 OPQR 10TN-274-2 EMS K326 (M₁) 0.935 MNOPQRSTU 10TN-283-2 EMS K326(M₁) 0.935 MNOPQRSTU 10TN-280-2 EMS K326 (M₁) 0.935 MNOPQRSTU 10TN-266-1EMS K326 (M₁) 0.935 MNOPQRSTU 10TN-288-2 EMS K326 (M₁) 0.934 MNOPQRSTU10TN-280-3 EMS K326 (M₁) 0.928 NOPQRSTUV 10TN-295-2 EMS K326 (M₁) 0.913OPQRSTUVW 10TN-292-2 EMS K326 (M₁) 0.913 OPQRSTUVW 10TN-295-4 EMS K326(M₁) 0.913 OPQRSTUVW 10TN-291-3 EMS K326 (M₁) 0.912 OPQRSTUVW 10TN-267-2EMS K326 (M₁) 0.899 QRSTUVW 10TN-287-1 EMS K326 (M₁) 0.892 QRSTUVWX10TN-295-3 EMS K326 (M₁) 0.889 QRSTUVWXY 10TN-281-4 EMS K326 (M₁) 0.888QRSTUVWXYZ 1oTN-279-1 EMS K326 (M₁) 0.880 RSTUVWXYZ[ 10TN-292-3 EMS K326(M₁) 0.877 STUVWXYZ[ 10TN-283-4 EMS K326 (M₁) 0.868 TUVWXYZ[ 10TN-266-4EMS K326 (M₁) 0.865 UVWXYZ[\ 10TN-273-2 EMS K326 (M₁) 0.862 UVWXYZ[\10TN-294-3 EMS K326 (M₁) 0.857 VWXYZ[\] 10TN-294-4 EMS K326 (M₁) 0.852WXYZ[\]{circumflex over ( )} 10TN-288-3 EMS K326 (M₁) 0.849WXYZ[\]{circumflex over ( )} 10TN-274-4 EMS K326 (M₁) 0.844WXYZ[\]{circumflex over ( )} 10TN-279-3 EMS K326 (M₁) 0.842WXYZ[\]{circumflex over ( )} 10TN-269-1 EMS K326 (M₁) 0.841WXYZ[\]{circumflex over ( )} 10TN-282-2 EMS K326 (M₁) 0.837XYZ[\]{circumflex over ( )}′ 10TN-280-4 EMS K326 (M₁) 0.837XYZ[\]{circumflex over ( )}′a 10TN-273-3 EMS K326 (M₁) 0.816YZ[\]{circumflex over ( )}′b 10TN-293-3 EMS K326 (M₁) 0.816Z[\]{circumflex over ( )}′ab 10TN-279-4 EMS K326 (M₁) 0.813[\]{circumflex over ( )}′abc 10TN-275-3 EMS K326 (M₁) 0.794\]{circumflex over ( )}′abcd 10TN-287-2 EMS K326 (M₁) 0.793\]{circumflex over ( )}′abcd 10TN-267-4 EMS K326 (M₁) 0.785 ]{circumflexover ( )}′abcde 10TN-275-1 EMS K326 (M₁) 0.784 ]{circumflex over( )}′abcdef 10TN-278-1 EMS K326 (M₁) 0.780 {circumflex over ( )}′abcdef10TN-295-1 EMS K326 (M₁) 0.770 ′abcdefg 10TN-284-1 EMS K326 (M₁) 0.767′abcdefgh 10TN-277-4 EMS K326 (M₁) 0.763 abcdefghi 10TN-283-3 EMS K326(M₁) 0.761 bcdefghi 1oTN-273-1 EMS K326 (M₁) 0.740 cdefghij 10TN-294-1EMS K326 (M₁) 0.738 defghij 10TN-286-3 EMS K326 (M₁) 0.736 defghij10TN-285-1 EMS K326 (M₁) 0.734 defghij 10TN-283-1 EMS K326 (M₁) 0.723defghijk 10TN-267-3 EMS K326 (M₁) 0.716 efghijk 10TN-271-2 EMS K326 (M₁)0.711 fghijkl 10TN-279-2 EMS K326 (M₁) 0.705 ghijklm 10TN-287-4 EMS K326(M₁) 0.694 hijldmn 10TN-269-4 EMS K326 (M₁) 0.691 ijldmn 10TN-278-3 EMSK326 (M₁) 0.680 jklinno 10TN-287-3 EMS K326 (M₁) 0.657 klinnop10TN-285-2 EMS K326 (M₁) 0.650 klinnop 10TN-278-2 EMS K326 (M₁) 0.640lrrmop 10TN-289-4 EMS K326 (M₁) 0.635 mnop 10TN-289-2 EMS K326 (M₁)0.622 nopq 10TN-273-4 EMS K326 (M₁) 0.614 opq 1oTN-271-1 EMS K326 (M₁)0.591 pqr 10TN-269-2 EMS K326 (M₁) 0.590 pq 10TN-275-2 EMS K326 (M₁)0.590 pq 10TN-278-4 EMS K326 (M₁) 0.588 pq 10TN-275-4 EMS K326 (M₁)0.551 qrs 10TN-289-3 EMS K326 (M₁) 0.514 rs 10TN-289-1 EMS K326 (M₁)0.511 rst 10TN-269-3 EMS K326 (M₁) 0.495 st 10TN-266-2 EMS K326 (M₁)0.444 t

Dark Air-Cured Results:

Forty-nine experimental dark air-cured M2 lines were evaluated againstNL Madole as the control (Table 3). Thus, Table 3 shows mean Cd contentin dark air-cured M2 breeding lines. For Table 3, values for the controlare shown in italics. Levels not connected by the same letter or symbolare significantly different. One line, 10TN-232, accumulatedsignificantly more Cd than NL Madole. Several of the lines (in bold)accumulated much less Cd than NL Madole control (shown in italics).

TABLE 3 Entry Pedigree/Name Mean Cd (μg/g) 10TN-232 EMS NL Madole (M₂)2.074 A 10TN-227 EMS NL Madole (M₂) 1.660 AB 10TN-226 EMS NL Madole (M₂)1.591 ABC 10TN-219 EMS NL Madole (M₂) 1.590 ABC 10TN-237 EMS NL Madole(M₂) 1.535 ABC 10TN-230 EMS NL Madole (M₂) 1.485 ABCD 10TN-222 EMS NLMadole (M₂) 1.455 BCD 10TN-235 EMS NL Madole (M₂) 1.398 BCD 10TN-236 EMSNL Madole (M₂) 1.369 BCDE 10TN-204 EMS NL Madole (M₂) 1.347 BCDEFG10TN-244 EMS NL Madole (M₂) 1.328 BCDEF 10TN-208 EMS NL Madole (M₂)1.324 BCDEFG 10TN-225 EMS NL Madole (M₂) 1.301 BCDEFG 10TN-214 EMS NLMadole (M₂) 1.286 BCDEFG 10TN-248 EMS NL Madole (M₂) 1.263 BCDEFG10TN-209 EMS NL Madole (M₂) 1.261 BCDEFG 10TN-238 EMS NL Madole (M₂)1.261 BCDEFG 10TN-200 NL Madole Control 1.251 BCDEFG 10TN-206 EMS NLMadole (M₂) 1.237 BCDEFG 10TN-241 EMS NL Madole (M₂) 1.234 BCDEFG10TN-249 EMS NL Madole (M₂) 1.218 BCDEFG 10TN-229 EMS NL Madole (M₂)1.196 BCDEFG 10TN-223 EMS NL Madole (M₂) 1.193 BCDEFG 10TN-215 EMS NLMadole (M₂) 1.164 BCDEFG 10TN-207 EMS NL Madole (M₂) 1.164 BCDEFG10TN-216 EMS NL Madole (M₂) 1.160 BCDEFG 10TN-202 EMS NL Madole (M₂)1.150 BCDEFG 10TN-234 EMS NL Madole (M₂) 1.148 BCDEFG 10TN-212 EMS NLMadole (M₂) 1.146 BCDEFG 10TN-213 EMS NL Madole (M₂) 1.120 BCDEFG10TN-210 EMS NL Madole (M₂) 1.110 BCDEFG 10TN-243 EMS NL Madole (M₂)1.091 BCDEFG 10TN-246 EMS NL Madole (M₂) 1.074 BCDEFG 10TN-240 EMS NLMadole (M₂) 1.071 CDEFG 10TN-247 EMS NL Madole (M₂) 1.059 CDEFG 10TN-220EMS NL Madole (M₂) 1.057 BCDEFG 10TN-211 EMS NL Madole (M₂) 1.044 BCDEFG10TN-242 EMS NL Madole (M₂) 1.037 BCDEFG 10TN-245 EMS NL Madole (M₂)1.034 CDEFG 10TN-250 EMS NL Madole (M₂) 0.992 DEFG 10TN-217 EMS NLMadole (M₂) 0.983 DEFG 10TN-221 EMS NL Madole (M₂) 0.959 DEFG 10TN-203EMS NL Madole (M₂) 0.956 CDEFG 10TN-233 EMS NL Madole (M₂) 0.919 DEFG10TN-239 EMS NL Madole (M₂) 0.899 BCDEFG 10TN-231 EMS NL Madole (M₂)0.891 DEFG 10TN-224 EMS NL Madole (M₂) 0.873 DEFG 10TN-201 EMS NL Madole(M₂) 0.805 FG 10TN-228 EMS NL Madole (M₂) 0.788 EFG 10TN-205 EMS NLMadole (M₂) 0.779 G

Twenty experimental dark air-cured M1 selections were evaluated againstNL Madole as the control (Table 4). Thus, Table 4 shows mean Cd contentin individual dark air-cured M1 breeding line selections. M1 lines withlowest and highest levels of Cd are shown in bold font; the control isshown in italics. Again, levels not connected by the same letter orsymbol are significantly different. Two M1 selections accumulatedsignificantly more Cd than control. Fourteen lines accumulatedsignificantly less Cd than control, but not less than the GOTHIATEK®standard of 0.51.1 μg/g.

As noted herein, the tobacco plants made by the methods of the inventionmay comprise at least one of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or10TN-287-4 lines described herein, wherein a representative sample ofseeds for these lines are deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209),under conditions prescribed by the Budapest Treaty, under ATCC AccessionNumbers PTA-12602, PTA-12601, PTA-12599 and PTA-12600, respectively.

TABLE 4 Entry Pedigree/Name Mean Cd (μg/g) 10TN-253-4 EMS NL Madole (M₁)1.494 A 10TN-252-4 EMS NL Madole (M₁) 1.471 A 10TN-254-4 EMS NL Madole(M₁) 1.290 B 10TN-200 NL Madole Control 1.251 B 10TN-254-3 EMS NL Madole(M₁) 1.248 BC 10TN-252-1 EMS NL Madole (M₁) 1.180 BCD 10TN-251-1 EMS NLMadole (M₁) 1.176 BCD 10TN-251_3 EMS NL Madole (M₁) 1.103 CDE 10TN-252-2EMS NL Madole (M₁) 1.072 DEF 10TN-255-1 EMS NL Madole (M₁) 1.068 DEF10TN-251-2 EMS NL Madole (M₁) 1.059 DEFG 10TN-255-2 EMS NL Madole (M₁)1.003 EFGH 10TN-253-1 EMS NL Madole (M₁) 1.001 EFGH 10TN-256-2 EMS NLMadole (M₁) 0.932 FGHI 10TN-254-2 EMS NL Madole (M₁) 0.930 FGHI10TN-257-1 EMS NL Madole (M₁) 0.912 GHI 10TN-253-3 EMS NL Madole (M₁)0.871 HI 10TN-254-1 EMS NL Madole (M₁) 0.871 HI 10TN-252-3 EMS NL Madole(M₁) 0.822 IJ 10TN-256-1 EMS NL Madole (M₁) 0.714 J 10TN-253-2 EMS NLMadole (M₁) 0.682 J

Example 3 Uptake and Distribution of Cd within the Tobacco PlantMutagenesis and Breeding

Seeds from K326, a flue-cured variety, were mutagenized with EMS (ethylmethane sulfonate) essentially as described for Example 1. The M0 mutantpopulations were screened on tissue culture media containing 150 μM Cd.Plantlets that survived the treatment were rescued and transferred tosoil. Self-seed was collected from each surviving plant in thegreenhouse. This M1 seed was sown and mature transplants transplanted tothe Tennessee field described in Example 2 (i.e., Table 1) known to havehigher than average Cd levels. These M1 flue-cured tobacco breedinglines were evaluated and screened under field conditions at thislocation. Early season samples were collected from lower stalk greenleaf and analyzed for Cd.

Based on early season (i.e., 65 days after transplant) heavy metal data,plants were selected for late season (i.e., 145 days after transplant)analysis to determine where the Cd was distributed, if at all. Lateseason samples were collected at about 145 days after transplanting fromhealthy plants. Lower, middle and upper lamina (no midribs) weresampled. Stalks, cut into three sections designated as lower, middle andupper, were also sampled. In addition, root and terminal bud sampleswere collected and analyzed for Cd content. All tissue was frozen on dryice and freeze-dried to maintain sample integrity. Freeze-dried sampleswere analyzed for Cd content. Three replicates of each sample wereanalyzed.

Cadmium Levels as Distributed Throughout the Plant:

Cadmium results from 13 early season experimental flue-cured M1 linesand K326 control are shown in Table 5. Thus, Table 5 shows Cd (pg/g)content of individual flue-cured M1 breeding line selections in planttissue. The Limit of Quantitation (LOQ) is 100 μg/g. Levels notconnected by the same letter or symbols are significantly different.

It can be seen that the K326 control accumulated the largestconcentration of Cd in upper lamina. Line 10TN-290-1 accumulated thelargest concentration of Cd in lower lamina tissue. Line 10TN290-2accumulated the largest concentration of Cd in middle lamina andflower/terminal bud tissues. Line 10TN-290-4 accumulated the largestconcentration of Cd in roots, lower stalk, middle stalk, and upper stalktissues. The lowest concentration of Cd in lower lamina tissue was foundin line 10TN-285-2. The lowest concentration of Cd in middle laminatissue was found in line 10TN-266-2. The lowest concentration of Cd inupper lamina tissue was found in line 10TN-278-4. The lowestconcentration of Cd in flower/terminal bud tissue was found in line10TN-278-6. Overall, root tissue accumulated the least amount of Cd inall samples including the control, K326. FIG. 2 shows the comparison ofbreeding lines evaluated. It was again found that the 10TN-290 M1 linesaccumulated the highest concentration of Cd compared to control.

TABLE 5 Flower/ Lower Middle Upper Terminal Entry Roots Lamina StalkLamina Stalk Lamina Stalk Bud 10TN- 0.191 AB 0.311 FG 0.132 B 0.166 I0.155 B 0.239 H 0.154 B 0.189 G 266-2 10TN- 0.133 CD n/a 0.117 BC 0.805B 0.132 C n/a 0.135 CD n/a 269-1 10TN- 0.105 DE 0.709 CDE <LOQ 0.485 F<LOQ 0.457 DE 0.102 FG 0.145 H 269-5 10TN- 0.091 E 0.593 DEF 0.115 BC0.679 CD 0.109 D 0.445 EF 0.121 DE 0.222 F 275-5 10TN- 0.109 CDE 0.348FG 0.054 D 0.267 H <LOQ 0.215 H 0.091 G 0.206 FG 278-4 10TN- 0.135 C0.515 EF <LOQ 0.488 F <LOQ 0.410 F <LOQ 0.212 FG 278-5 10TN- <LOQ 0.495EFG 0.053 D 0.410 G <LOQ 0.329 G <LOQ 0.144 H 278-6 10TN- 0.100 CDE0.215 G 0.124 BC 0.268 H 0.128 C 0.317 G 0.146 BC 0.363 C 285-2 10TN-0.102 DE 0.439 EFG 0.118 BC 0.511 F 0.127 C 0.520 C 0.148 BC 0.513 A285-5 10TN- 0.093 E 1.287 A 0.111 C 0.718 CD 0.133 C 0.721 AB 0.149 BC0.456 B 290-1 10TN- 0.106 CDE 1.068 AB 0.119 BC 0.945 A 0.127 C 0.685 B0.162 B 0.517 A 290-2 10TN- 0.093 E 0.854 BC n/a 0.664 DE 0.104 D 0.491CD 0.111 EF 0.314 D 290-3 10TN- 0.205 A 0.848 BCD 0.255 A 0.748 BC 0.321A 0.501 C 0.344 A 0.335 D 290-4 K326 0.165 B 0.852 BC 0.124 BC 0.607 E0.155 B 0.731 A 0.157B 0.267 E Control

Table 6 shows the ratio of root to above ground tobacco tissues for Cdaccumulation for late season tobacco (i.e., 145 days aftertransplanting) as tobacco tissue [Cd]/root [Cd]). It can be seen thatwhen the distribution of μg Cd per gram (g) dry weight in upper tobaccotissues versus roots was expressed as a ratio, the 10TN-290-1,10TN-290-2, and 10TN-290-3 lines had the highest ratio of Cd in alllamina positions. This suggests that these lines hyper-accumulate Cd tolamina compared to control. Conversely, 10TN-266-2 had the lowest ratioof Cd in all lamina positions even though it had a higher concentrationof Cd in its roots. This suggests that the Cd is not translocated tolamina as efficiently as the 10TN-290 lines. Additionally, K326 had thehighest ratio of Cd in the upper stalk position and 10TN-285-5 had thehighest ratio of Cd in the flower/terminal bud tissue.

It can be seen that the 10TN-266-2 line does not move Cd from roots toabove ground tissue as efficiently as other lines evaluated. This can bea desirable trait in that the leaves will have less Cd. Lines such as10TN-290 M1, which hyper-accumulate Cd, may be used to remove Cd fromthe soil (i.e., for field clean-up).

Thus, these results show the ability to alter the distribution of Cdwithin the plant through traditional plant breeding methods.

TABLE 6 Flower/ Termi- Lower Middle Upper nal Entry Lamina Stalk LaminaStalk Lamina Stalk Bud K326 5.16 0.75 3.68 0.95 4.43 2.44 1.62 Control10TN-290-2 10.08 1.12 8.92 1.2 6.46 1.53 4.88 10TN-290-1 13.84 1.19 7.721.43 7.75 1.6 4.9 10TN-290-4 4.14 1.24 3.65 1.57 2.44 1.68 1.6310TN-290-3 9.18 n/a 7.14 1.12 5.28 1.19 3.38 10TN-285-5 4.3 1.16 5.011.25 5.1 1.45 5.03 10TN-275-5 6.52 1.26 7.46 1.2 4.89 1.33 2.4410TN-269-5 6.75 n/a 4.62 n/a 4.35 0.97 1.38 10TN-278-5 3.81 n/a 3.61 n/a3.04 n/a 1.57 10TN-285-2 2.15 1.24 2.68 1.28 3.17 1.46 3.63 10TN-266-21.63 0.69 0.87 0.81 1.25 0.81 0.99 10TN-278-6 n/a n/a n/a n/a n/a n/an/a 10TN-269-1 n/a 0.88 6.05 0.99 n/a 1.02 n/a 10TN-278-4 3.19 0.5 2.45n/a 1.97 0.83 1.89 The root sample for 10TN-278-6 was <LOQ and thereforea ratio is not reported. The samples in bold accumulated a high ratio ofCd given availability of Cd in roots compared to K326.

Thus, EMS mutation breeding techniques were successful in identifyingtwo flue-cured M₁ breeding lines that fall below the GOTHIATEK® limitfor Cd. Also, in certain embodiments, genes from wild species can beintrogressed into modern tobacco as an alternate genetic source forreduction in Cd uptake.

Thus, the methods of the present invention utilize a rapid andeconomical mutagenesis method for developing plant lines having asignificantly altered uptake and/or altered levels of at least oneenvironmental contaminant or other non-natural chemical. The alterationin uptake and/or levels may be a reduction in the uptake and/or levelsof the environmental contaminant or other non-natural chemical. Or, thealteration in uptake and/or levels may be an increase in the uptakeand/or levels of the environmental contaminant or other non-naturalchemical. In an embodiment, the plant is tobacco. The invention providesa method whereby mutagenized tobacco seeds are allowed to germinateunder selective conditions, and then chimeric plants at least partiallycomprising a phenotype resistant to high concentrations of cadmium oranother environmental contaminant or non-natural chemical are selected.

Included in the present invention are modified tobacco lines having analteration in a heavy metal. The heavy metal having altered uptakeand/or altered levels may be at least one of arsenic (As), cadmium (Cd),chromium (Cr), nickel (Ni), lead (Pb), selenium (Se), zinc (Zn), copper(Cu), mercury (Hg) or silver (Ag). In an embodiment, the heavy metalhaving altered uptake and/or levels is cadmium. Or, other heavy metalsand/or other selected elements may be altered. These geneticallymodified tobacco lines can be used as germplasm to develop new tobaccovarieties with altered heavy metal profiles and/or can be mixed withother strains of tobacco to produce a blend having improved taste andaroma.

All references cited herein are incorporated by reference in theirentirety.

What is claimed is:
 1. A method for producing a modified tobacco plantcomprising generating a tobacco plant comprising significantly altereduptake and/or altered levels of environmental contaminant or othernon-natural chemicals as compared to an unmodified tobacco plant fromwhich the modified plant is derived.
 2. The method of claim 1 comprisingthe steps of: incubating at least one tobacco seed from an unmodifiedtobacco plant in a solution comprising a mutagen; washing the at leastone seed free of the mutagen; germinating the at least one seed andgrowing at least one M0 tobacco seedling in the presence of a selectionagent to generate at least one M0 tobacco plant comprising M1 tobaccoseeds, wherein the M1 tobacco seeds from the chimeric tobacco plantcomprise at least one mutagenized M1 tobacco seed; and germinating theat least one mutagenized M1 tobacco seed to select for a modified M1tobacco plant comprising significantly altered uptake and/or alteredlevels of environmental contaminant or other non-natural chemicals ascompared to the unmodified tobacco plant.
 3. The method of claim 2,wherein the selection agent is a heavy metal.
 4. The method of claim 2,wherein the selection agent is cadmium.
 5. The method of claim 1,wherein the environmental contaminant or other non-natural chemicalhaving altered uptake and/or altered levels is at least one of arsenic(As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), selenium(Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag).
 6. The methodof claim 1, wherein the environmental contaminant or other non-naturalchemical having altered uptake and/or levels is cadmium.
 7. The methodof claim 1, wherein the modified tobacco plant comprises at least a 40%reduction in cadmium levels.
 8. The method of claim 1, wherein themodified tobacco plant is the species Nicotiana tabacum.
 9. The methodof claim 1, further comprising the step of growing a plurality of themodified M1 tobacco plants to generate M2 tobacco seeds, and germinatingthe M2 seeds to grow M2 plants, wherein at least one of the M2 tobaccoplants is a homozygote for a mutation conferring the ability to grow inthe presence of the selection agent.
 10. A modified tobacco plantcomprising a significantly altered uptake and/or altered levels ofenvironmental contaminant or other non-natural chemicals as compared toan unmodified tobacco plant from which the modified plant is derived,wherein the genome of the modified tobacco plant comprises a mutationthat exhibits a dominant phenotype of resistance to growth in thepresence of a selection agent.
 11. The modified tobacco plant of claim10, wherein the selection agent is a heavy metal.
 12. The modifiedtobacco plant of claim 10, wherein the selection agent is cadmium. 13.The modified tobacco plant of claim 10, wherein the environmentalcontaminant or other non-natural chemical heavy metal having altereduptake and/or altered levels is at least one of arsenic (As), cadmium(Cd), chromium (Cr), nickel (Ni), lead (Pb), selenium (Se), zinc (Zn),copper (Cu), mercury (Hg) or silver (Ag).
 14. The modified tobacco plantof claim 10, wherein the environmental contaminant or other non-naturalchemical having altered uptake and/or levels is cadmium.
 15. Themodified tobacco plant of claim 10, wherein the modified tobacco plantcomprises at least a 40% reduction in cadmium levels.
 16. The modifiedtobacco plant of claim 10, wherein the modified tobacco plant comprisesthe species Nicotiana tabacum.
 17. The modified tobacco plant of claim10, where the plant is made by a method comprising the steps of:incubating at least one tobacco seed from an unmodified tobacco plant ina solution comprising a mutagen; washing the at least one seed free ofthe mutagen; germinating the at least one seed and growing at least oneM0 tobacco seedlings in the presence of a selection agent to generate atleast one M0 tobacco plant comprising M1 tobacco seeds, wherein the M1tobacco seeds from the chimeric tobacco plant comprise at least onemutagenized M1 tobacco seed; and germinating the at least onemutagenized M1 tobacco seed to select for a modified M1 tobacco plantcomprising significantly altered uptake and/or altered levels ofenvironmental contaminant or other non-natural chemicals as compared tothe unmodified tobacco plant.
 18. The modified tobacco plant of claim17, further comprising the step of growing a plurality of the modifiedM1 tobacco plants to generate M2 tobacco seeds and further germinatingthe M2 seed to grow M2 plants, wherein at least one of the M2 tobaccoplants is a homozygote for a mutation conferring the ability to grow inthe presence of the selection agent.
 19. The modified tobacco plant ofclaim 10, comprising at least one of the 10TN-278-2, 10TN-253-4,10TN-256-1 or 10TN-287-4 lines.
 20. A tobacco plant comprising at leastone of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines.
 21. Atobacco product comprising a modified tobacco having significantlyaltered uptake and/or altered levels of an environmental contaminantand/or a non-natural compound as compared to an unmodified tobacco fromwhich the modified tobacco is derived.
 22. The tobacco product of claim21, wherein the environmental contaminant and/or other non-naturalcompound having altered uptake and/or altered levels is at least one ofarsenic (As), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb),selenium (Se), zinc (Zn), copper (Cu), mercury (Hg) or silver (Ag). 23.The tobacco product of claim 21, wherein the environmental contaminantand/or other non-natural compound having reduced uptake and/or levels iscadmium.
 24. The tobacco product of claim 21, wherein the modifiedtobacco plant comprises at least a 40% reduction in cadmium levels. 25.The tobacco product of claim 21, wherein the modified tobacco plant isthe species Nicotiana tabacum.
 26. The tobacco product of claim 21,comprising at least one of the 10TN-278-2, 10TN-253-4, 10TN-256-1 or10TN-287-4 lines.
 27. A composition comprising tobacco from at least oneof the 10TN-278-2, 10TN-253-4, 10TN-256-1 or 10TN-287-4 lines.